Recent clinical trials have substantiated hydroxyurea (HU) to be a potent effector of hemoglobin F (HbF) production in patients with sickle cell disease. This augmentation in HbF levels in patients is associated with a decrease in relative beta chain synthesis and thus an inhibitory effect on hemoglobin S (HbS) polymerization both through the decrease in MCH (S)C and the specific sparing effect of HbF. In order to gain insight into the possible genetic mechanisms underlying these effects, we used cation exchange (CE) and reverse-phase high performance liquid chromatography (HPLC) to examine changes in the levels of each hemoglobin species. Samples from ten patients treated with HU were analyzed approximately twice each week. Within the first 50 days of HU treatment, HbF increased on average from 3.3% to 7%. The patients had an average initial ratio of G- gamma/A-gamma of 0.72 which did not change with treatment. At baseline, approximately 15-20% of the fetal hemoglobin exists in the acetylated form (F(1)) as determined by CE-HPLC, independent of subtype. With HU treatment, the total amount of F(o) and F(1)) increases roughly proportional to the increase in F-reticulocyte numbers. Although the total HbA(2) level appears unaffected by treatment, there is an increase in the proportion of one modified HbS species, which may co-migrate with Hb(2), after about 50 days of treatment. For three patients treated with a combination HU and erythropoietin, we found a further increase in both F(1) and this modified HbS fraction to levels approaching 5% and 10% respectively. Subsequent analysis of this modified beta-globin polypeptide showed that the NH2 was not blocked (Edman sequencing) and that the modifier had a mass of 300 close to the one of glutathione (307) (mass spectroscopic analysis). A dual strategy using deblocking and synthetic methods has been used followed by IEF analysis and confirmed the glutathione nature of the adduct. Chemically modified HbS may interfere with HbS polymerization and thus have an added clinical benefit. Acetylation of other proteins (e.g., histone) may be involved in the regulation of gene expression; naturally occurring glutathione adducts have only been reported very recently. It is possible that their formation is related to metabolic effects of HU treatment or, as with acetylation, this modification may be indicative of induced fetal erythropoiesis.